PROTEINS.

STRUCTURE AND FUNCTION

Lecture 7
 Supersecondary structures (domain organisation)

= structural motifs = folds = particularly stable arrangements of several

elements of secondary structure and the connections between them
– helix-turn-helix
– leucine zipper
– calcium binding turn, found between two helices
– zinc finger
– Greek key
– β-α-β

Greek key motif

β-α-β motif β hair pin motif αα motif

 Leucine zipper Zinc finger

The „motif“ term is used in the structural biology under two aspects:
1. The term defines a particular sequence of amino acids, characteristic
for a specific biological function. Such an example is the so called motif “zinc
finger”, found in a family of various proteins, which bind DNA.
 2. “motif“ means a group of adjacent elements of the secondary
structure that has a particular functional significance, or it defines a
portion of a domain, independently folded.
Examples:
– the motif of the 2-helix bundle, found to proteins that bind DNA.
– the motif of the 4-helix bundle (found at many hormones and other types of
proteins);
– the “Rossmann folding”, an α/β fold that binds NAD+ cofactors and has clear
functional implications.
– the “catalytic triad” of serine-proteases, made of Asp, His and Ser residues,
which interact between them and which can be situated in different positions,
as a function of the protease family in which they appear.

• These amino acid residues form a catalytic unity with identical geometry
and with the same biochemical function.

• In each class, different arrangements of these elements are possible, and

each distinct arrangement is a structural motif.
α Motifs
• The motif of the 4-helix bundle – is found in a large variety of α domains
and has different functions: oxygen transport, binding nucleic acids, and
electron transport. In humans is found mostly in the growth hormone.
• The motif - “globin fold”, found in myoglobin, and which contains 8 α-
helices forming inside of the domain a hydrophobic “pocket” where
organic and organo-metallic groups can bind. The 8 helices form a single
domain that wraps up the heme. This motif appears in a different form in
cytochroms too, proteins involved in electron transport.

β Motifs
• formed only of antiparallel β structures, connected by β turns and large
bends. Proteins that are formed of β motives are: immunoglobulins,
certain enzymes as superoxid dismutase, and proteins that bind
carbohydrates at the cell surface.
• I.e. the enzyme neuraminidase of the influenza virus is formed of a
repetitive motif of 4 antiparallel chains.
B. Tertiary Structure

• refers to the spatial arrangement of amino

acid residues that are far apart in the
sequence and to the pattern of disulfide
bonds. It includes the arrangement of β-pleated sheet
different secondary structures.
• Secondary and tertiary structures induce
stereospecificity, responsible of the chemical
and biological reactivity of proteins.

• Domain represents the basic fundamental unit

of the three-dimensional structure;
• it is a distinct entity in terms of conformation
and function.

• Protein conformation depends on the protein

primary structure and is characterised by the
alternation of regulated and rigid structures
(α helix and β-pleated sheet) with
disordered, flexible parts, apparently
randomly folded.
 Structural domains
• The structural motifs can associate to form supersecondary,
stable, three-dimentional structures, called domains (proteic
modules).

• Domains are distinct entities as conformation and biological

properties, being unitarily maintained in the protein structure by
the intermeddle of chain fragments with non-regular structure.

• Some proteins, as keratine in hair, are fibrous, but most of the

proteins are globular, their peptide chains wrapping to realise a
compact form.

• MW = 1,000 - 1,000,000 Da.

• MW < 20,000 ⇒ simple globular form, with the diameter 20-30

Å,

• bigger proteins form, usually, two or more structural domains.

 DOMAINS

• usually, but not always, are

formed of a continuous
segment of amino acid
sequences

• are capable to form a

structure, with own stability
in water solutions.

• These structural domains

often keep a part of the
biochemical function of the
big protein they derived
from.
 DOMAINS:
• Generally, a domain has 100-
250 amino acids, having a
hydrophobic core and a
hydrophilic exterior.
• The hydrophobic cores are
essential for domains stability.
• So far, the highest number of
domains found in a protein is
13.
• Domains associate in a protein
by the same interactions that
stabilize the internal structure
of the protein.
• The folding process is the transition from primary structure to the final form
(native state) of a protein
• The folding takes place spontaneously due to the bonds that are formed
between the side chains of amino acid residues.
• These bonds are mainly hydrophobic, but in the case of certain proteins
hydrogen bonds, ionic, ester bonds, disulfide bridges –S-S- between Cys
residues can also be formed. The latter have a strong stabilized structure:
insulin, hair keratin, digestive enzymes.
• the structure of each protein is unique
• X-rays cristallography and NMR methods were used to reveal
the structure of thousands of proteins

MIOGLOBIN STRUCTURE : A. BALL AND STICK MODEL b. Ribbon model

MIOGLOBIN STRUCTURE : distribution of AA
• it has been found that different proteins (with different primary structure,
different functions or different species) present a high similitude concerning the
arrangement of the supersecondary structures. Such examples are:
– The model with all the domains having an α-helix structure, in which 7 or 8 α-
helix segments are bound by small peptide segments that allow their folding
in a globular form. The model is found to lysozime, myoglobin, hemoglobin
subunits.
– The model of domains α-β in which the domains with α structure bind to
domains with β structure which allows the adoption of a barrel type
conformations (triosephosphate isomerase) or twisted paper (lactate
dehydrogenase).
– The model with all domains having β structure, in which segments with β
strand structure are bound together by small peptide segments that allow
their folding in a globular form. The model is found to superoxide dismutase,
immunoglobulins, concanavalin A

immunoglobulin and Protein G

 HEMOGLOBIN

LYSOSYME
 LACTATE DEHYDROGENASE

TRIOSEPHOSPHATE
ISOMERASE
 IMMUNOGLOBULINS

SUPEROXIDE
DISMUTASE
• There are many proteins with two or more structural identical
domains. I.e. thioesterase from E. coli

• These proteins with a similar structure develop by gene

duplication. It is supposed that a single gene, which encodes a
domain of this kind of protein, is sequential copied so the two
genes will fusion, and their sequences will express a single
polypeptide.

• Sometime the gene duplication can appear in a single

structural domain, as it happens in the case of proteins γ-
crystalline in humans and β-crystalline in mouse.

• Conclusion = the protein formed of many structural domains

develops by gene fusion, genes that have previously
encoded distinct proteins.
According to the tertiary structure, proteins are divided into two categories:
• Globular, which have α helix and β sheet segments that alternate with
loops or β turns. They are divided into two subclasses:
– intracellular -soluble ones and
– Membrane- insoluble ones (intramembrane channels).
• I.e. ribonuclease has 124 amino acid residues, 4 disulfide bridges, an α
helix domain and an antiparallel β sheet.
• Fibrous, where a type of secondary structure is extended on a large
portion of the molecule. The high content in Gly, Pro, and HO-Pro makes
them insoluble.
• They have a structural localization.
Primary

Secondary

Tertiary

quaternary
 C. Quaternary Structure

• Generated by the interactions between independent

polypeptide chains
• is characteristic to globular proteins with molecular weights
higher than 60,000 Da and present an oligomeric structure.
• The oligomeric assemble contains several monomeric subunits
(protomers), identical or different, bound together by weak, van
der Waals, ionic or hydrogen type bonds.
• ⇒ the quaternary structure is controlled by the protein primary
structure, which encodes the place of contact between
protomers, within the oligomeric structure.
• Ex. - hemoglobin molecule • Ex. –rhinovirus
• Hemoglobin is functional • Four types of subunits
only in the conditions of
tetrameric arrangements
 the structure is very labile, being easily disorganised at
dilution, pH variations, adding of concentrated urea,
guanidinium chloride solutions, etc.
If the oligomeric assemble is reversibly disorganised under
the action of a denaturant agent, when this agent is removed
from the medium, the quaternary structure is completely re-
established.
Christian Anfinsen work (1950)
on enzyme ribonuclease
COOPERATIVE FOLDING OF PROTEINS
• Protein folding / unfolding : “all or none” process that
results from a cooperative transition.
• Conditions that lead to the disruption of any part of a
protein structure are likely to unravel the protein
completely.
• The structural properties of proteins provide a clear
rationale for the cooperative transition.
• Structures that are partly intact and partly disrupted are not
thermodynamically stable and exist only transiently.
• Cooperative folding ensures that partly folded structures
that might interfere with processes within cells do not
accumulate.
• How does the protein make the transition
from the unfolded state to the unique
conformation of the native (folded) state?
• How long should take this random search?
• 5x 1047 conformations 1.6x1027 years

Proteins fold by progressive stabilization of

intermediates rather than the random serach
• The essence of protein folding is the retention of partially correct
intermediates

• The total free energy difference between folded/unfolded state of

a protein composed of 300AA is 10 kcal/mole → the contribution
of each AA to the energy required to maintain folded state is very
small → the correct intermediates can be lost easily

• The interactions that lead to the cooperative folding can stabilize

intermediates as structure builds up.

• Local regions with significant structural preference can adopt

their favored structures (not necessary stable on their own) and,
as they form can interact one other leading to increased
stabilization
 Dynamic aspects of protein structure
NMR determinations, fluorescence, X-ray diffraction have
shown that atoms in a protein do not exist in a static
position, but rather have dynamic, fluid movements.
X-ray diffraction shows that in very well packed structures
there are small folding “defects”, which conduct to the
apparition of “holes” or small spaces that permit a
flexibility of molecule atoms.
Dynamic behaviour of proteins is implied in conformational
changing, induced by substrate, inhibitors, allosteric
effects, electron transfer.
Existence of conformation is due to activity of special
proteins in the cell, chaperone proteins that, during the
synthesis of a protein, guide the formation of conformation
while preventing their interaction with various factors that
would shift to another type of protein conformation
 MODIFICATIONS OF PROTEINS CONFORMATION

• Many proteins are covalently modified, through the attachment of groups

other than amino acids, to modify their functions:
•
– Acetyl groups are attached to the amino termini of many proteins, a
modification that makes these proteins more resistant to degradation.
– The addition of hydroxyl groups to many proline residues stabilizes fibers of
newly synthesized collagen, a fibrous protein found in connective tissue and
bone.
– γ-Carboxyglutamate formation in blood clotting factors
– Many proteins (on the surfaces of cells or are secreted) acquire carbohydrate
units on specific asparagine residues. The addition of sugars makes the
proteins more hydrophilic and able to participate in interactions with other
proteins.
– The addition of a fatty acid to an α-amino group or a cysteine sulfhydryl group
produces a more hydrophobic protein.
– the phosphorylation of the hydroxyl amino acids serine and threonine in - The
phosphoryl groups on these three modified amino acids are readily removed;
thus they are able to act as reversible “switches” in regulating cellular
processes.
– Many proteins are cleaved and trimmed after synthesis (digestive enzymes,
blood clotting factors, polypeptide hormones, virus proteins).
 Allostery

• (allos = different, steros = body, structure) represents the modification of

protein conformation under the action of external chemical factors
(allosteric effectors).
•
• They bind to specific sites (allosteric site) and produce a modification of
protein conformation.

• The new conformation can favour the biological activity (positive effector,
or activator) or they can have an inhibiting effect (negative effector or
inhibitor).

• Allostery is reversible; the effector removal makes the protein to regain its
initial conformation.
General allosteric mechanism
 Structural base of protein functions

• Protein functions are determined by their three-dimensional structure.

Understanding function through structure is a primary goal of structural
biology, but this fact is not facile because the definition of function itself
can be seen in several respects: biochemical, physiologic, cellular, genetic,
etc.
• An example in this regard is tubulin, a protein that has many functions at
different levels:
• In terms of biochemistry, tubulin is an enzyme with role in GTP hydrolysis,
but also a structural protein that gives rise by polymerisation to
protofilaments and microtubules;
• At the cell level, tubulin forms a lattice of microtubules that starts at the
centrosome level, and has a role in the transport of different components
(proteins, vesicles, etc) from one part of a the cell to another;
• In dividing cells, tubulin has a role in the formation of mitotic axis that
divides the chromosome into the two daughter cells;
• In some mobile eukaryotic cells, tubulin forms cills and flageli with role in
locomotion.
• In general, protein function is easily deduced by studying the sequence
and structure
• in some cases supplementary investigations are needed.
• In genomic age, function can be determined by comparative analysis of
sequences at genome level and by recognition of functional motifs in the
primary and tertiary structure.

• There are four main functions of proteins:

– establishment of interactions with various functional elements;
– catalytic role;
– “switching” role
– structural role
 Regulation mechanisms of protein function

• density of protein molecules in a cell is very high

• protein molecules are separated by very small distances, which may
contain only a few water molecules
• Regulation of protein function in vivo - different mechanisms:
a) localization of gene products and/or of species they interact with;
b) covalent or noncovalent binding of effector molecules;
c) by the amount and lifetime of active proteins.

a) Localisation - Not all proteins have absolute specificity ⇒ multiple

functions.
Activity regulation of such enzymes is achieved by:
- ensuring the presence of their active form only in those
compartments where necessary (signal-sequences)
- their binding to other macromolecules (lipids, carbohydrates,
protein), with the formation of complexes involved in achieving of a
specific function.
– When the protein is not properly located, it is maintained in an
inactive conformation.
 b) Effectors binding - induce conformational modifications, having as
result the passing of the protein in an active or inactive form.

• Effectors - small dimensions (proton), large dimensions (macromolecules)

and can be bound covalently or noncovalently, modifying reversibly or
irreversibly the protein structure. For example, in enzymes, these effectors
can bind:
– at the level of the active site and are called competitive inhibitors,
– in another zone then the active site, these compounds being called allosteric
effectors, and the enzyme is an oligomeric and allosteric enzyme.

• Allosteric enzymes have several binding sites for different ligands. The
binding of effector molecules to the allosteric enzymes is characterised by
cooperativity; this represents the influence that an effector has (when
binds to a binding site) on other binding sites. Cooperative effect can be:
– positive (activator) – when the binding of a ligand molecule at the level of a
site determines the conformational modification of the protein, so the binding
of other ligands is favoured;
– negative (inhibitor) – when the binding of a certain ligand to a site has an
inhibitor role on the binding of other ligands.
• Effector binding can be covalent, or can produce covalent modification of the protein,
as follows:
– phosphorylation
– covalent binding of lipids and carbohydrates
– methylations of side chains
– acetylation of aminoterminal groups
– proteolytic splitting of the polypeptide chain at one or several sites.
• more than 40 covalent posttranslational modifications have been identified; most
majorities determine the modification of the settlement of a protein, its activity, or its
interaction with other proteins and macromolecules.

• Limited proteolysis occurs through the proteolytic cascade in the amplification of low
concentrations of some transitory signals.

• In proteolytic cascade, an initial stimulus activates a proteolytic enzyme; this one, in

turn, activating the following enzymes involved in that cascade.
For example:
– complement cascade – activated as a response to a microbial agent, having role in
immune defence;
– blood coagulating cascade – involved in stopping the bleeding of various causes.
•The most common form of covalent modification is the reversible
phosphorylation at serine, threonine and tyrosine level.

kinase

phosphatase
 c) Activity of a protein can be controlled, also, by controlling its
quantity and life time in the cell.
- quantity of protein – at transcription level, controlled by: -
nature of promoter,
- action of transcription factors (activators or repressor)
- variation of RNAm degradation
- Protein lifetime is determined by:
- speed of degradation
- varies a lot function of protein
A single protein can be regulated in different ways
 Proteins with multiple functions
Protein Function Supplementary function
Glucose-6-phosphate-
Glycolytic enzyme Cytokine
isomerase
EF-1 Elongation factor in translation Protein that binds actin
Peptidyl-prolyl cis-trans
cyclophilin Regulates calcineurin
isomerase
MIF(macrophage Inhibitor of macrophages and
Phenyl-pyruvate isomerase
inhibitory factor ) lymphocytes T
PutA Proline-dehydrogenase Repressor of transcription
Maintains SH groups in reduced DNA polymerase subunit
Thioredoxin
form T7 phag
Ligand for a certain type of
Thrombin Protease – in blood clotting
receptor on cell surface
Thymidylate-
Enzyme in nucleotide synthesis Inhibitor of translation
synthetase
Protein –type “chaperone“ in
FtsH Metallo-protease
bacteria
LON Mithocondrial protease Protein-type “chaperone”
Methionine Protects eIF2 of
Peptidase
aminopeptidase 2 phosphorylation